The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative has the ambitious goal of elucidating how neuronal ensembles interactively encode higher brain processes. To accomplish this goal, new and improved methods for both recording and manipulating neuronal activity will be needed. In this application, we focus on technologies for manipulating neuronal activity. The major significance of this application is that we will provide an enhanced chemogenetic toolbox that allows non-invasive, multiplexed spatiotemporal control of neuronal activity in domains ranging from single synapses to ensembles of neurons. To achieve this, we will provide: Chemical actuators with improved pharmacokinetics and pharmacodyamics suited for use with current DREADDs in eukaryotes ranging from Drosophila to primates (Specific Aim #1) Photo-caged CNO and other chemical actuators to provide millisecond-scale control (Specific Aim #1) Novel DREADDs and 'split-DREADDs' targeted to distinct neuronal pathways to enable multiplexed interrogation of neuronal circuits (Specific Aims #2 and 3) Chemogenetic platforms with minimized desensitization and down-regulation (Specific Aim #3)